Journal of Muscle Research and Cell Motility 3, 113-126 (1982)

Abstracts of the Tenth European Conference on Muscle and Motility G A L W A Y , 1 5 - 1 7 S E P T E M B E R 1981

MUSCLE GROWTH,

DEVELOPME NT AND DISEASE

Control of protein metabolism and growth i n muscles of the rat D a v i d F. G o l d s p i n k

Department of Physiology, The Queen's University of Belfast, U.K. In most mammals approximately 20-25% of the body weight and protein content at birth can be attributed to skeletal muscle. By the time the adult state has been reached this value is nearer 45%, through both a larger increase in muscle mass relative to other body tissues, and a greater protein composition within the individual muscles. During the postnatal growth of muscle, the rates of protein synthesis and protein breakdown decrease with age, while at the same time coming closer together to reach a steady state in the mature, non-growing animal. That is, in the rat the fractional rate of synthesis decreases from 12-20% at three weeks (precise values depend upon the muscle type studied) to 4-9% at ten months. Similarly, the fractional rate of breakdown falls from 5-10% to 3-8% over the same period. In contrast, other tissues such as the liver and thymus do not show the same changes with age. The influence of physiological factors such as (1) hormones (e.g., insulin and the glucocorticoids), (2) mechanical signals (e.g., contractile activity and passive stretch) and (3) exercise was discussed in relation to their effects on protein turnover and hence muscle growth.

Genetic influences on muscle growth A. C. B. H o o p e r

Department of Anatomy, University College,Dublin, Ireland Muscle mass is determined by a large n u m b e r of genes acting at many loci. Quantitative genetic methods provide a useful model for the study of muscle growth and may assist animal breeders to increase the meat yield of farm animals. Both the n u m b e r and dimensions of the fibres are influenced 0142-4319/82/010113-14504.06/0

by genetic factors. Fibre n u m b e r accounts for two thirds of the alterations in muscle mass following selection for altered body weight, while fibre diameter accounts for slightly more than half of the remainder and fibre length for slightly less than half. Studies of inbreeding depression, maternal effects and heterosis suggest that additive genetic effects are more important in the determination of fibre n u m b e r and indicate a high heritability for this parameter. Genetically determined differences in fibre length are due to changes in the n u m b e r of sarcomeres along the fibres. The lengths of the sarcomeres and of the actin and myosin filaments are not affected by artificial selection, although there are species differences in myofilament length. Selection for altered body weight does not affect the cross-sectional area of myofibrils and differences in fibre n u m b e r are due to changes in myofibril number. Quantitative genetic control of the mass of muscles is due to the determination of the n u m b e r of their cellular and sub-cellular functional units. The mechanisms through which this control is mediated are not known.

The relation between ATPase activity, SDH activity and cross-sectional area of skeletal muscle fibres in mice W. J. v a n d e r L a a r s e a n d P. C. D i e g e n b a c h

Zoological Laboratory, University of Amsterdam, Amsterdam, The Netherlands From histological sections we have derived some quantitative parameters which concern contractile properties of muscle fibres. A modification of the histochemical method for the demonstration of myosin ATPase described by Meijer [Histochemie 22, 51-58 (1970)] allowed a quantification of relative ATPase activities of muscle fibres in one section. We used this technique to investigate the relation between ATPase and SDH activity in mice skeletal muscle (m.soleus, m.plantaris and re.extensor digitorum longus).

© 1982 C h a p m a n a n d H a l l Ltd.

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Muscle growth,

SDH activity has been quantitated as described by Pool et al. [Histochemistry 64, 251-262 (1979)]. Subsarcolemmal mitochondria have not been included in the measurements. Fibre cross-sectional area has also been determined. The SO and FG fibre types show a small variation in the examined parameters, while the FOG fibres show a large variation in fibre cross-sectional area as well as SDH activity, the smallest fibres having the highest SDH. The ATPase activity of FG fibres is slightly higher than the ATPase activity of FOG fibres, although, on the basis of this parameter as well as the SDH activity and cross-sectional area, no distinct groups between the fast fibres can be found.

parvalbumin anti-sera were identified as neurons. Astro- and oligodendroglia remained unstained. Parvalbumin was found to be a neuronal marker protein for a distinct subpopulation of neurons scattered throughout the central nervous system. In the cerebellum, Purkinje cells showed strongest immunoreactivity, whereas the granule cells displayed no staining. In all parvalbumin-positive neurons, soma, dendrites and axons were homogenously labelled, whereas nuclei remained unreactive. The distribution of calmodulin and S-100 in the central nervous system is different. In neurons, parvalbumin may be functionally involved in axonal flow, which is known to be Ca 2+ dependent, but other roles (release of neurotransmitters) cannot be excluded. In the muscle, parvalbumin was exclusively located in the type II (fast twitch) fibres and not in type I (slow-twitch oxidative) fibres. The type II fibres could be further subdivided into at least five subgroups displaying distinct staining intensities. Our results support the hypothesis that parvalbumin in r~pscle is concerned with its fast relaxation and also indicates a graduation in the relaxing properties of muscle fibres belonging to the same histochemical type.

Parvalbumin, a neuronal marker protein in rat brain: microisolation and comparison with the muscle protein M a r t i n W. Berchtold a n d Claus W. H e i z m a n n

Institut fiir Pharmakologie und Biochemie der Universitiit Ziirich, Switzerland Parvalbumins, highly specific calcium receptor proteins, have only been isolated and characterized from muscle tissues, although an immunological crossreactivity has also been found in brain. This may be mostly because brain has a significantly lower concentration of parvalbumin and conventional isolation procedures cannot be applied. In view of our latest immunohistochemical results, demonstrating that parvalbumin in the central nervous system of the rat is a neuronal marker protein, a biochemical characterization of the brain parvalbumin has been performed. For this purpose a new HPLC-method was applied, allowing isolation of parvalbumin from crude tissue extracts containing low concentrations (in brain cortex approximately I mg kg-1). Parvalbumin, well separated from S-100, TN-C and calmodufin, was eluted as a single protein peak from a C18 reverse phase column (applying an acetonitril or 2-propanol gradient in a phosphate/perchlorate buffer). Parvalbumin from rat muscle was isolated and its primary sequence determined (FEBS Lett., in press). The direct comparison of the muscle and the brain component revealed identities in their migrations on 2D-gels, HPLC-elution profiles and immunological properties. These results indicate similar (if not identical) structures for muscle and brain parvalbumin but the immunohistochemical results indicate distinct functions.

Parvalbumin may have distinct functions in brain and muscle C l a u s W. H e i z m a n n a n d Marco R. Celio

Institut fiir Pharmakologie/Biochemie und Anatomisches Institut der Universitiit Ziirich, Switzerland Parvalbumin was localized in the rat central nervous system and in the muscle tissue by means of immunohistochemical techniques (Nature, Lond., in press). All cells in the brain immunostained by the

development

and disease

Glucocorticoid action on the growth of four muscles in the rat F. J. Kelly a n d D. F. G o l d s p i n k

Department of Physiology, The Queen's University of Belfast, U.K. The growth response of different body tissues, including muscle, is known to vary considerably after exposure to high circulating levels of glucocorticoid hormones. In this study we have considered the action of a potent synthetic glucocorticoid, dexamethasone, on four differing muscle types: a fasttwitch skeletal muscle (tibialis anterior), a slowtwitch skeletal muscle (soleus), cardiac muscle and smooth muscle (the sclerosa of the small intestine). The heart increased in size, while the growth of the soleus was arrested after five days of the steroid administration (2.5 mg kg -~ day-l). In marked contrast, over the same time, wasting occurred in both the tibialis anterior and the sclerosa. The induced changes in growth will be explained by the steroid's action on protein turnover in each muscle type, with induced changes in protein breakdown clearly playing a prominent role in modifying their growth patterns. In vitro effects of synthetic and naturally occurring glucocorticoids on muscle protein turnover J o s e p h i n e A. M c G r a t h a n d D. F. G o l d s p i n k

Department of Physiology, The Queen's University of Belfast, U.K. The in vivo administration of glucocorticoid steroids is known to markedly alter plasma levels of other hor-

Muscle growth, development and disease

115

mones, e.g. insulin. Such endoc~ne changes may indirectly mediate the profound alterations of muscle protein turnover which we previously observed following steroid treatments. In the present study, a direct action of glucocorticoids on skeletal muscle has been confirmed. Inhibition of both protein synthesis and protein breakdown was observed in the isolated soleus muscle following in vitro exposure ~o either synthetic or naturally occurring glucocorticoids. The latency of steroid action on protein turnover was also investigated. At in vitro steroid concentrations of 10 -6 M, synthetic steroids (dexamethasone, trimacinolone) were no more effective than the natural hormones (cortisol, corticosterone) in producing changes in protein turnover. This is in contrast to our earlier findings that synthetic steroids were much more potent when administered in vivo. This apparent discrepancy could be related to the complete saturation of the muscles' steroid receptors at in vitro concentrations of 10 -6 M, thereby masking any potency differences between the steroids. The in vitro responses of protein synthesis and breakdown to cortisol and dexamethasone were still similar at 10 -8 M. At lower concentrations, the response to cortisol was lost while dexamethasone remained effective at 10 - l ° M, thereby establishing its greater potency both in vitro and in vivo.

circulation; therefore, the mince must contain the cells responsible for the regeneration. In a third series of experiments, the mince was split into two halves before the grafting procedure. One of the halves was frozen in liquid N 2 and thawed. It was combined with the remaining hall and the mixture was orthotopically grafted. The regenerates were contractile: 30 days after the surgery, their tetanic isometric force was 8 -+ 1 g, i.e. one fourth of that developed by regenerate originating from an untreated mince. The tetanic isometric force increased at 60 days (22 + 5g) and 90 days (58 ± 13 g) but remained much lower than that observed in regenerates arising from untreated minces. These regenerates contract rather slowly, their B constant being 6 -+ 1, 8 + 2 and 16 -+ 3 m m s -1 respectively 30, 60 and 90 days after surgery. These data indicate that the tetanic isometric force and the B constant of the force-velocity relation increase steadily during the regeneration, at least up to 90 days after surgery. The rate of these increases is slowed if half of the mince is frozen and thawed before grafting. Growth of muscles regenerating from a mince seems thus to depend on the initial number of cells living in the mince.

Muscle growth during regeneration after mincing E. G h i n s a n d G. M a r e c h a l U.C.L., Av. Hippocrate, B-1200 Bruxelles, Belgium Rat gastrocnemius muscles were minced and orthotopically grafted. The tetanic isometric force of the regenerating muscle was 33 ± 5 g (n = 3) 30 clays after the surgery. It increased linearly afterwards, being 69 ± 11 g at 60 days and 107 -+ 10 g at 90 days (this is about one tenth of the force given by a normal gastrocnemius). The force-velocity relationship was measured in all the regenerates, and was characterized by a B constant quite low at 30 days, 13 + 3 m m s -1. It is increased to 19 + 2 mm s -1 at 60 days and 20 ± 1 at 90 days. Thus, even after 90 days of regneration, the muscles were rather slow, their B constant being half that of normal gastrocnemius. They were however still much faster than true slow muscles such as soleus (whose B constant is about 8 mm s-l). We have tried to obtain some information on the source of the cells responsible for the regeneration in the following way. If the mince is frozen in liquid N 2 and thawed just before the grafting procedure, the regenerates are very small and they cannot be stimulated (n = 12). Histologic examination showed that they are completely devoid of muscle cells. The freezing procedure kills all the cells that were spared by the mincing. The absence of muscle regeneration indicates that the mince is not invaded by myoblast brought by the

An approach to establishing the percentages of histochemical fibre types in skeletal muscle M. J. Clancy a n d Janet Lester Muscle Biology Laboratory, An Foras Taluntais, Dunsinea Research Centre, Castleknock, Co. Dublin, Ireland Satisfactory histochemical typing of myofibres involves an acceptable nomenclature, dependable histochemical methods and valid sampling procedures, especially of cryosections. A widely accepted classification recognises: (1) fl-Red (fiR) myofibres, which possess, (a) an acid-stable, alkali-labile myosin ATPase, (b) a high oxidative capacity, as indicated by succinate dehydrogenase, and (c) a low to moderate glycolytic activity as indicated by glycogen phosphorylase staining; (2) o-red (oR) myofibres, which possess an acid-labile, alkali-stable myosin ATPase, and with moderate to high oxidative and glycolytic activities; and (3) o-white (~W) myofibres possessing an acidlabile, alkali-stable myosin ATPase, with low oxidative and high glycolytic capacities. Considerable improvements in staining have now been made by using media with a standard ionic strength of 0.16 M, a pH optimum for reaction, and high buffering capacity. The precision in identifying oR myofibres was improved by combining the SDH reaction with (a) the acid-stable myosin ATPase on one cryosection, and with (b) the alkali-stable myosin ATPase on a second. The identifications of the three myofibre types were achieved with these combinations and by correlations with other histo-

116

Muscle growth, development and disease

chemical reactions (glycogen phosphorylase and a-glycerophosphate oxidoreductase). The fasciculus was established as a suitable sample unit for enumerating the myofibre types. Following examination of variation of the ratio of the three myofibre types between fascicular areas in the cryosections, the percentage of the different myofibre types is computed over all the areas enumerated.

evidence for myofibre abnormality was found in any of the samples examined. This report is the first to record that endogenous and exogenous anabolic agents affect significantly the proportions and sizes of muscle fibre types in a meat animal.

The effects of anabolic compounds on the fibres of bovine L. dorsi muscle M. J. Clancy, Janet M. Lester a n d J. F. R o c h e

An Foras Taluntais, Animal Production Research Centre, Dublin, Ireland Anabolic compounds are used in meat animal production to increase growth rate, feed conversion, and the lean meat content of the carcass. While their value for meat production is well established, nothing is known about the effects of these substances on the cellular aspects of muscle growth and development. Samples of longissimus muscle were taken from carcasses of steers, steers implanted with Ralgro and Finaplix, and bulls of Friesian and Charolais cross Friesian breeds of cattle. Percentage and mean cross-sectional areas of fl-red (fiR), co-red (~R), and a-white (~W) muscle fbres were determined. While implanted steers had the same percentage fiR myofibres as the untreated steers, they had 21% more aR and 11% less ~W myofibres. These effects were accompanied by hypertrophy of both the fiR and aR myofibres in the treated steers, while the size of the ~W myofibres were unchanged. While bulls had the same amount of fiR myofibres as treated steers, they had 22% more ~R and 23% less ~W myofibres than the implanted steers. Significant hypertrophy of all three myofibre types occurred in bulls. A significant breed difference was observed in the case of the percentage fiR, ~R, and ~W myofibres, and in the sizes of the fiR and ~R myofibres. No

Actin and myosin content in atrophying and regenerating muscle A. Jakubiec-Puka, K. Krajewski, D. K u l e s z a - L i p k a a n d J. K o r d o w s k a

Nencki Institute of Experimental Biology, Warsaw, Poland The content of contractile proteins actin and myosin was evaluated in the atrophying and regenerating rat soleus muscle. Atrophy of the muscle or its regeneration following reinnervation were produced by cutting or crushing of the sciatic nerve, respectively. The amounts of actin and myosin heavy chains (myosin HC) were measured in myofibrils and in muscle protein extracts obtained quantitatively. In the atrophying muscle a decrease of the myosin HC to actin ratio takes place; it was observed as early as on the third day after denervation. This disproportion of the ratio appeared as the result of more intensive lowering of the myosin content in the muscle than that of actin. The amount of actin decreased in parallel to the decrease of the atrophying muscle weight. In the early stage of regeneration (on the 14th to 21st day after crushing the nerve) a rapid increase of the myosin content in the muscle took place, causing reduction of the disproportion of myosin HC to actin ratio. This disproportion, however, remained till about the 35th day after crushing the nerve. The changes observed in the ultrastructure of the contractile apparatus are in agreement with the conclusions from the biochemical investigations. The results suggest that, under pathological conditions or functional requirements of muscle, myosin filaments are more labile than other components of contractile apparatus.

CONTRACTILE PROTEINS Structure of rabbit skeletal myosin in solution: electric birefringence, static and dynamic light scattering studies R. C a r d i n a u d 1 a n d J. C. B e m e n g o 2

1Service de Biophysique, D~partement de Biologie, CEN Saclay, 91191 Gif-sur- Yvette Cfdex, France ;Laboratoire de Biophysique, Universitf de Nice, Parc Valrose, 06000 Nice, France Myosin and HMM solutions have been studied in high ionic strength media by means of static and dynamic light scattering and electric birefrigence techniques. Homodyne measurements of the laser light scattered from myosin solutions manifested a characteristic D value dependence on myosin and phosphate concentrations. Using the typical D versus (myosin) curves obtained in the presence of 0.5 M

phosphate and 0.2M phosphate respectively a's references it was shown that: (1) the observed phenomena are completely reversible; (2) minor components such as C protein and F protein do not significantly influence the measured D values; (3) the effect of preparation procedures on these dynamic light scattering measurements is negligible. Contrary to translational parameters, rotary diffusion constants of myosin and HMM were found to be independent of protein concentration and depend only slightly on the phosphate concentration of the buffer. The electric polarizability of both molecules is mainly due to a permanent dipole moment. From these results, five out of six association models proposed by Harrington & Burke [Biochemistry 11, 1448-55 (1972)J were excluded and if myosin forms

117

Contractile proteins dimers they can be of the side to side parallel type only. From the hydrodynamic parameters the dimensions and flexibility of myosin and H M M molecules are discussed particularly in relation with one of the Garcia de la Torre and Bloomfield model [Biochemistry 19, 5118-23 (1980)] which best fits our results.

The birefringence of smooth muscle, its change during contraction A n n e G o d f r a i n d - d e Becker a n d J. M. Gillis

Physiology Department, University of Louvain, Belgium As birefringence measurements may give information on the packing and orderly arrangement of molecules, this approach was chosen to investigate whether or not smooth muscle would change its optical properties from rest to contraction. Anococcygeus muscles of the rat were stimulated with noradrenaline (NA); tension (P), thickness (d) and optical retardation (F) were measured. The ratio F/d gives the birefrinsence of the muscle (B). At rest, B was 0.883 x 10-5(+0.053 x 10-3, n = 27), andd, 254/~m. When maximum isometric tension (Prnax) was developed (NA: 10-5-10 -4 M), B was increased by +0.424 x 10 -3 (-+0.048 x 10 -3, n = 40). This was merely due to a change of F (+ 30%) combined with a decrease of d (-12%). In muscles extracted with Triton X 100 and kept in a relaxing medium, B was 0.705 x 10 -3 (-+0.094 x 10 -3, n = 4). Contracting solutions evoked a mild contraction ( - 0 . 2 Pmax) and a birefringence change of + 0 . 4 8 0 x 10 3 (-+0.048 x 10 -3, n = 8) with AF + 39% and Ad - 11%. The maximal change of B in fresh and extracted muscles is the same in absolute value. It is thus not due to stress nor to membrane signals, and seems related to the contractile mechanism; it may reveal the formation of myosin filaments on activation.

A simulation study of the distribution of Ca and Mg between troponin, parvalbumins and the sarcoplasmic reticulum, at rest and during contraction J. M. Gillis a n d J. L e f e v r e

Physiology Department, University of Louvain, Belgium The model is composed of (1) T sites ('Ca specific'), present in troponin only: 0.14raM; (2) P sites ('Ca-Mg sites') present both in troponin and parvalbumins (variable concentration), and (3) the Ca p u m p of the SR, operating according to Michaelis kinetics (Km: 10 7 M Ca2+; Vmax: 700/~mol Ca s - 1 1 - 1 of living muscle). 1. Re sting state: equilibrium between the various complexes of the ligands for steady concentrations of Ca 2+ (2.5 x 10-~M) and Mg 2+ (3 raM) (in %): Tsites: apo-form 65, T-Mg: 30; T-Ca: 5;P sites : apo: 2, P - M g : 88, P-Ca: 10.2. Kinetics of contraction: Changes in the concentration of the complexes which results from an instantaneous pulse of Ca (200 #tool 1-1), and its eventual removal by the SR pump. The system has five non-linear differential equations, solved by the iteration method. The rate

constants used come from Potter et al. [In Calcium Structure & Function, Elsevier (1980)], It is found that: (1) 97% Ca saturation of T sites is obtained in a few milliseconds, in spite of high concentration of P sites (0.84 raM, frog muscle); (2) Relaxation of T-Ca is caused by transfer of Ca to P sites and capture by the SR; (3) Ca picked up by P sites returns eventually into the SR; (4) w h e n the SR activity is reduced, the contribution of P sites to relaxation is much more important.

binding Protein,

Cytochalasin-induced ATPase activity of actin Peter Dancker

Max-Planck-Institut fiir medizinische Forschung, D-69 Heidelberg, West Germany During polymerization of actin, cytochalasin B (CB)induced ATPase activity is proportional to the concentration of unpolymerized actin. When polymerization velocity is augmented (e.g. at higher Mg concentration), the initial ATPase activity is also augmented but reaches its low steady-state value earlier than w h e n polymerization velocity is slower. Of the two possible explanations for CB-induced ATPase activity (increased exchange between free and filament-bound monomers or property of monomers) we favour the view that the monomers themselves are the true enzyme. Otherwise ATPase activity should depend also on the concentration of filament ends (and should be no simple linear function of monomer concentration) and one had further to assume that CB increases the 'flow' of monomers through each filament; this, however, is not supported by current evidence. Therefore, we think, as Brenner & Korn do []. biol. Chem. 255, 841-4 (1980)] that CB uncouples an ATPase activity of monomers which is normally associated with polymerization, from polymerization.

Isolation and contraction of obliquely striated muscle cells from the body wall of Lumbricus terrestris J. D ' H a e s e a n d A. D i t g e n s

Institut fiir Zoologie H der Universitiit Dfisseldorf, Universitfitsstr. 1, 4000 Diisseldorf,, West Germany Isolated cells and myofibrils have proved to be useful 'model systems' to study the organization and contraction of vertebrate cross-striated and smooth muscles. In the present study, cells were dispersed from the extended earthworm body ~vall by incubation with collagenase (1 m g m1-1) in a modified Hanks' salt solution. The length of the ribbon-shaped muscle cells was variable and often exceeded 1 ram. On the addition of ATP, the muscle cells contracted to about 30% of their original length without a marked increase in diameter. The time needed for full contraction of the cells varied depending on the conditions and was in m a n y cases only a few seconds. Experiments using EGTA together with MgATP followed by the addition of Ca demonstrated the Casensitivity of the cell contraction. After glycerination

118 of the cell preparations the oblique striation was more pronounced and the cells became more fragile and tended to splice into fibrils. Thin sections of glycerinated cells revealed that the Z-rod material was extracted and contractile fibrils were formed running obliquely to the cell axis. No evident difference was found in the contraction behaviour of glycerinated and freshly prepared muscle cells, suggesting that shearing between filaments of adjacent 'sarcomers' might be the main contraction process. (Supported by Stiftung Volkswagenwerk.)

Biochemical properties of native myosin filaments T. K. C h i n a n d A. J. R o w e

Department of Biochemistry, University of Leicester, Leicester LE1 7RH, U.K. Chymotryptic digestion of synthetic myosin filaments produces S-1 lacking in DTNB LC [Weeds & Taylor, Nature, Lond. 257, 54 (1975); Weeds & Pope, J. molec. Biol. 111, 129 (1977)]. W e have now shown, however, that chymotryptic digestion of D20-H20 gradients-purified native myosin filaments [Emes & Rowe, Biochim. Biophys. Acta 537, 195 (1978)] yields an S-1 population containing the DTNB LC (Chin & Rowe, in press). Our results suggest that this difference between synthetic and native myosin filaments when subjected to chymotrypfic digestion is due to the difference in the myosin conformation within the two types of filaments after exposure to high ionic strength in the case of synthetic myosin filaments. Using an isotopic assay ([?-32p]ATP), detailed studies of the ATPase activities of purified native myosin filaments and the derived S-1 (CT) have been made, before and after exposure to high ionic strength. It is shown that DTNB LC before exposure to high ionic strength possesses the potential to repress ATPase activities [Chin & Rowe, unpublished results)]. It is believed that DTNB LC plays a role in the 'switching off' of ATPase activity in relaxed muscle. Using myofibrils with or without the troponin-tropomvosin system, Lehman [Biochem. J. 163, 291 (1977); Nature, Lond. 274, 80 (1978)] has shown that the ATPase activities of rabbit skeletal muscle were Ca2+-sensitive at physiological ionic strength. Using purified myosin filaments and relaxed filament preparations, we have shown that these preparations have Ca2+-sensitive ATPase activities both at physiological and low ionic strengths in the case of purified native myosin filaments. Our results indicate that DTNB LC which has not been exposed to high ionic strength media, is responsible for Ca 2+sensitivity in vertebrate skeletal myosin ATPase activities.

Some type-specific proteins in single muscle fibres of the cat A. R o w l e r s o n , U. Reist, H. H o w a l d and E. J e n n y

Institut fiir PharmakoIogie und Biochemie der Vet. Med.

Contractile proteins

Fakultfit der Universitfit Ziirich, Winterthurerstr. 260, CH-8057 Ziirich, Switzerland; and Forschungsinstitut ETS, CH-2532 Magglingen, Switzerland An electrophoretic analysis of the protein composition of single muscle fibres of four different histochemical fibres types was made, using the methods described by Billeter et al. [Eur. J. Biochem. 116, 389-95 (1981)]. The results obtained show that: (1) The distribution of myosin LC within cat fibre types I, lia and lib follows the pattern previously found in human and rabbit muscle; (2) all IIm fibres have two LC, both different from the fast and slow LC of the other fibre types; (3) cat type I fibres from both pure and mixed muscles contain both forms of LCls (i.e. LClsa, LClsb) previously described for myosin extracted from soleus (pure type I) muscle; (4) type IIm fibres contain a non-myofibrillar protein which is never seen in type I, lIa or IIb fibres; and (5) I-D peptide mapping of myosin HC from fibre types I, Ha, IIb and lim shows that these are all different, the difference being greatest for IIm compared to the other three, and least for IIa compared to IIb. A.R. is grateful to E.M.B.O. for financial support, and to Dr C. Heizmann for advice and discussion.

31p n.m.r, investigations of phosphoserine groups in bovine cardiac troponin in dependence of Mg 2+ and Ca 2+ ions N. Beier 1, K. D. S c h n a c k e r z 2 a n d L. M. G. H e i l m e y e r , Jr 1

iInstitut fiir Physiologische Chemie, Lehrstuhl I, Ruhr-Universit~it, 4630 Bochum 1, Postfach 10 21 48 West Germany 2physiologisch-Chemisches Institut der Universit~it Wiirzburg, Koelliker-Strasse 2, 8700 W~irzburg, West Germany Bovine cardiac troponin (cTn) was isolated by the LiC1 extraction method of Tsukui & Ebashi [J. Biochem., Tokyo, 73, 1119-21 (1973)] with a final purification step on a DEAE-cellulose column. Troponin, rims prepared, was homogeneous according to SDSPAGE. The endogeneous phosphate content of cTn varied considerably, ranging from 0.0 to 1.8 Pi per 100 000 of molecular weight. In one half of a cTn preparation, having a phosphate content of 0.1 mol P i per TnT subunit, the phosphate was removed with calf intestine alkaline phosphatase. The 31p Fourier-transformed n.m.r, spectra of both parts were recorded as described by Sperling et aL [Eur. J. Biochem. 101, 581-92 (1979)]. Only the untreated sample showed a 31p resonance signal, which could be identified as a freely rotating phosphoserine by its chemical shift (& - 4.46 to - 4.24) at pH values from 7.15-7.05 and the line width of the signal2+•(2.0-4.0 Hz). Successive addition" of Mg 2+ and Ca Ions up to concentrations of 10 mM and 2 mM, respectively, did not cause a significant change in the chemical shift or the line width. A change would be expected, if the phosphate group is influenced by the

119

Muscle membranes binding of the metal ions. This serine phosphate seems to be comparable to the phosphate group at serine-1 of the TnT subunit of rabbit skeletal muscle [Sperling et al., Eur. J. Biochem. 101, 581-92 (1979)]. Moreover, the untreated and the phosphate-free

MUSCLE

samples were both phosphorylatea at the TnIsubunit by cAMP-dependent protein-kinase. The divalent metal ions dependent changes of the 31p_ resonance-signals will be discussed.

MEMBRANES

Skeletal muscle microsomal membranes D. R. H e a d o n

Department of Biochemistry, University College, Galway, Ireland Rabbit skeletal muscle contains three well-defined membrane systems namely, the plasma membrane, transverse tubule membranes and the intracellular membranes of the sarcoplasmic reticulum. Indeed, the sarcoplasmic reticulum is itself morphologically differentiated into three distinct regions: the terminal cisternae, longitudinal elements and the central fenestrated collar. Biochemical differentiation has been reported. Homogenization of skeletal muscle results in vesiculation of the membranes giving rise to membranes which are isolated in the microsomal fraction. Subsequent density gradient centrifugation resolves this fraction into heavy and light components. The amount of heavy and light component present is a function of the homogenization conditions. Physical, enzymic and lipid compositional differences are evident between the heavy and light subfractions. Labelling studies indicate that the larger and lighter membranes are of external origin. These membranes (median density of 1.15 g m1-1) have a high cholesterol content and display a high basal ATPase activity. In contrast the smaller and heavier microsomal membranes (median density 1.18) are not labelled under conditions which label external membranes, are essentially devoid of cholesterol and display a high level of CaATPase activity. Protein analysis by SDS polyacrylamide gel electrophoresis and particularly by isoelectric focusing show substantial differences between these subfractions.

Molecular interactions regulating calcium movements across the sarcoplasmic reticulum P. J. Q u i n n

Department of Biochemistry, ChelseaCollege, University of London, Manresa Road, London, U.K Ca 2+-ATPase of sarcoplasmic reticulum is an intrinsic membrane protein that depends upon surrounding membrane lipids or suitable detergents for its biochemical activity. Studies have been undertaken to examine the role of membrane lipids in the function of the enzyme and in particular to determine the origin of discontinuities in Arrhenius plot of enzyme activity. Modulation of the fluidity of sarcoplasmic reticulum membranes by cholesterol incorporation, phospholipid exchange and homogeneous catalytic hydrogenation of unsaturated membrane lipids has

indicated that the activity of Ca2+-ATPase is directly related to membrane fluidity. Changes in membrane fluidity, however, do not appear to alter the temperature at which discontinuities in Arrhenius plots of enzyme activity are observed. These effects appear to be due to a thermally induced conformational change in the protein which is associated with an uncoupling of ATPase from the net accumulation of calcium inside the vesicles. Uncoupling can be retarded by preparing membranes in the presence of the thiol reducing agent, dithiothreitol, or calcium ions. The present results do not support the idea of a lipid annulus surrouhding the intrinsic membrane protein which regulates el~zyme activity but rather they emphasize the integrated role of the enzyme in creating and possibly dissipating, the gradient of calcium ions across the sarcoplasmic reticulum.

Mechanism of the action of estradiol on heart tissue Evert L. de Beer

Department of Physiology, University of Utrecht, The Netherlands The female sex hormone estradiol acts both on the electrical and mechanical activity of heart tissue [de Beer et aL, In Cardiac Dynamics: Models and Measurements, New York, Plenum (1981)]. The mechanism of the action of estradiol (up to 200 pg m1-1) was studied by a combination of electrical and mechanical tools. Action potentials of atrial tissues were studied after the administration of estradiol. Ionic blockers (Verapamil and CsC12) were added to the perfusion fluid and the effect of estradiol was reinvestigated. These experiments suggest that estradiol diminishes mainly the slow inward current. However, when the effect of estradiol on the mechanical activity of the isolated left ventricle was studied, the isovolumic pressure development exhibits another view. The rate of pressure development was enhanced, while the time to reach peak pressure was decreased. This observation, combined with the electro-physiological ones, suggests, that estradiol accelerates the slow inward current.

The sodium ion dependence of muscle protein metabolism S h e e n a E. M. L e w i s a n d D. F. G o l d s p i n k

Department of Physiology, The Queen's University of Belfast, U.K. The influence of external sodium on in vitro rates of amino acid transport and protein turnover of the

120 extensor digitorum longus (EDL) muscles of rats (50 g) has been investigated. In skeletal muscle, neutral amino acids are transported by at least two specific carrier systems, the 'A' and 'L' systems. We have investigated the influence of Na + on these transport systems using three separate amino acids; ~-aminoisobutyric acid (i.e., AIB) representing the 'A' system, tyrosine carried by the 'L' system and cycloleucine possessing affinities for both systems. Rates of amino acid influx and efflux were measured. A1B influx was inhibited and its effiux enhanced upon depletion of Na +. These changes resulted in reduced accumulation of AIB in the EDL, with this effect becoming more pronounced with progressive depletions of external Na +. Net transport of cycloleucine was also significantly inhibited but to a lesser extent than AIB. All components of tyrosine transport were unaffected by the reduction of external Na +. The transport of amino acids into muscle is linked with the synthesis of n e w proteins. Since amino acid transport is influenced by the external Na + concentration, we further examined the influence of this ion on protein turnover. Decreasing external Na + progressively inhibited protein synthesis while enhancing protein breakdown thus giving rise to a catabolic state in the muscle.

Occluded bound magnesium on the phosphorylated intermediate of the sarcoplasmic transport ATPase Madoka Makinose

Max-Planck-Institute for Medical Research, Department of Physiology, Heidelberg, West Germany Magnesium is an essential factor for the reversible reaction process of the SR Ca-pump and substituted by M n [Makinose & Hasselbach, Pflfigers Archiv. 274, $7 (1961)]. Recent kinetic studies have s h o w n that two Mg 2+ are needed for the forward phosphorylation of the SR transport ATPase (one as the direct activator and the other as Mg-ATP, i.e. true substrate) [Makinose & Boll, Cation Flux Across Biomembranes, N e w York, Academic Press, and Func-

Muscle membranes their stability and the occluded bound Mg 2+ besides the occluded b o u n d Ca 2+ reported already [Takisawa & Makinose, Nature, Lond. 290, 271 (1981)].

Cholesterol, phospholipid and basal ATPase activity M a r y A. N. M o l l o y a n d D e n i s R. H e a d o n

Department of Biochemistry, University College, Galway, Ireland Treatment of the microsomal fraction of skeletal muscle with Triton X-100 and sodium deoxycholate at detergent to protein ratios of 5.0 and 0.5 (mg m g - 1), respectively, considerably altered the ATPase activities of the fraction. 99.6% of the calciumdependent ATPase activity was solubilized. Basal ATPase activity was not solubilized to the same extent, the maximum value being 32%. However, the basal ATPase activity appears to be reduced/inhibited to 1% of the control value. Associated with this alteration was the solubflization of 98.5% of the membrane cholesterol and 98.7% of the m e m b r a n e phospholipid. [3H]Cholesterol was added in the form of a sonicated aqueous suspension to a detergent-treated particulate sample. The microsomes bound exogeneous cholesterol in greater quantities than that present in the control. Associated with this cholesterol binding was the apparent reactivation of the basal ATPase activity from 0.005/~mol Pi rag- ~ min-1 in detergenttreated samples to 0.34~mol Pi mg -1 min -1 in samples containing 2.5 ~mol exogeneous cholesterol mg protein-1, an apparent reactivation to 68% of the basal ATPase activity present in the control. Phosphatidyl choline was bound in amounts up to 750 ~g phospholipid mg protein -1. Reincorporation of phospholipid resulted in a restoration of basal ATPase activity to 20% of the control value.

The influence of Triton X-100 on the physical properties of skeletal muscle microsomes O r l a M. C o n n e e l y a n d D e n i s R. H e a d o n

tion and Molecular Aspects of Biomembrane Transport,

Department of Biochemistry, University College, Galway, Ireland

Amsterdam, Elsevier/North-Holland (1979)]. For the backward phosphorylation, only one Mg 2+ is needed [Kolassa et al., FEBS Lett. 108, 495 (1979)]. Using 54Mn as the Mg-analogue Triton-treated SR vesicles were phosphorylated and isolated rapidly from the soluble components in assay by the method of Penefsky [Penefsky, J. biol. Chem. 252, 2891 (1977)]. The amount of transferred 32po 4 was compared with that of bound 54Mn. The EP formed in the forward reaction (EPv) binds equimolar 54Mn in an occluded form independent of the energy supplying substrate. The EP formed in the backward phosphorylation (EPh) cannot be detected after the Penefsky method and seems to be more unstable than EP v. Apparently, the spedes of the phosphorylated intermediate of the SR ATPase can be characterized chemically by

The microsomal fraction of skeletal muscle tissue may be fractionated on sucrose density gradients into a light and heavy subfraction. Cholesterol and basal or Ca-independent ATPase activity are associated with vesicles of the light subfraction whereas the heavy subfraction contains a high Ca-dependent ATPase activity and is essentially devoid of cholesterol [Headon et al., Molec. Cell. Biochem. 17, 117-23 (1977)]. Studies on the influence of Triton X-100 on the physical properties of the microsomal fraction indicate variations in the susceptibility of the light and heavy subfractions to solubilization, the heavy membranes being more susceptible. Treatment of microsomes with Triton X-100 results in a decrease in the phospholipid to protein ratio of the membranes from

Muscle mechanisms and energetics 590/~g mg -1 in control microsomes to 40 ~g mg -1 after treatment at a detergent to protein ratio of 5. This decrease is reflected in an increase in the density of treated m e m b r a n e s w h e n fractionated on sucrose density gradients ranging from 1.04 to 1.22 g m1-1. The difference in modal density between control and Triton-treated microsomes is 0.066, while the median density increases by 0.049, indicating that the membranes have not all been solubilized to the same degree. The difference in susceptibility of the light and heavy subfractions to solubilization by Triton X-100 reflects differences in the susceptibility of basal ATPase and Ca-dependent ATPase containing membranes to solubilization and is possibly due to differences in the fluidity of these m e m b r a n e s [Lowe & Coleman, Biochem. Biophys. Acta 640, 55-65 (1981)].

Calorimetric studies of two ATP driven transducing systems of muscle Takao Kodama

Department of Physiology, University College, London, U.K.

MUSCLE MECHANISMS

121 The hydrolysis of ATP cata!ysed by sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle was studied by microcalorimetry in parallel with measuring the extent of hydrolysis and the level of the phosphorylated intermediate ( E - P) of the Ca-ATPase in the reaction medium. During the first few turnovers of the enzyme catalytic cycle, w h e n an appreciable amount of E - P is still present, the observed heat is much smaller than the heat expected from the amount of ATP hydrolysed. Since the accompanying Ca uptake, inclusive of Ca binding to internal sites, is associated with very little heat change [Kodoma et al., J. Biochem., Tokyo, 88, 1259-65 (1980)], this result indicates that the formation of E - P is endothermic and hence driven by a larger increase in entropy. The overall exothermicity of the ATP hydrolysis is brought about by the subsequent, strongly exothermic, decomposition of E - P. Thus, there are certain similarities in the enthalpy diagram between the mechanisms of the Ca-ATPase and the myosin ATPase [cf. Kodama & Woledge, J. biol. Chem. 254, 6382-6 (1979); Kodama, J. biol. Chem. 256, 2928-33 (1981)].

AND ENERGETICS

Isometric contraction of fast and s l o w rat muscles at different lengths and temperatures exposed to octanoate F. K 6 s s l e r a n d G. Kfichler

Central Institute of Occupational Medicine, Department of Work Physiology, 1134 Berlin, East Germany Previous investigations concerning the effect of fatty acids on frog muscles were extended to mammalian muscles. The influence of sodium octanoate (0.5-5mM) on isometric twitch and tetanus of isolated m. soleus (Sot.) and m. extensor digitorum longus (EDL) of rats at different length and temperature was studied. The length-tension relation of both muscles was tested in the range of 10+-6mm. Octanoate did not change this relation. The active developed tension following stimulation was depressed in relation to the concentration and time of exposure by 10-60%. The time to peak and the half relaxation time were shortened by about 30% in both EDL and Sol. In relation to the tension developed at different length of muscles octanoate caused a proportional depression of twitch and tetanus. There was no significant shift of the maximum of the tension development. At constant temperature the twitch/ tetanus ratio was not altered by octanoate. In relation to the temperature (4-40 ° C), the twitch and tetanus amplitudes, the latency, the time to peak, and the half relaxation time were analyzed and compared for fast and slow muscles in Ringer's solution and in octanoate.

Ventricular filling: an inotropic intervention P. S c h i e r e c k a n d P. J. M. Kil

State University of Utrecht, Department of Physiology, Vondellaan 24, Utrecht, The Netherlands In the classical view of muscle mechanics, 'contractility' (e.g. Vmax) was independent of the rest length of the muscle. However, nowadays there is strong evidence that rest length is an important factor in the release of activator Ca from the SR. In intact heart preparations this indicates that the way of left ventricular filling influences the contraction phase, not only based on changes of the myofibrillar overlap of the fibres within the wall. Applying quick volume changes in diastole just before stimulation causes a shift of the classic Starling relation, comparable with that induced by changes in Ca 2+ of the perfusate. This is supported by stiffness measurements in systole, relating stiffness (dP/dV) with pressure at the time at which stiffness is measured. The linear stiffness-pressure relation rotates if very slow volume changes are applied in diastole and shifts along itself if quick changes are applied in diastole. These properties can be interpreted in terms of a rest length • dependent Ca 2 + -reflux across the sarcolemma of heart muscle. (Supported by Grant 79.105 from the Netherlands Heart Foundation.)

122 The decay of the active state of the inferior oblique muscle of the rabbit: influence of temperature and denervation G. A s m u s s e n

Carl-Ludwig Institute of Physiology, Karl Marx University, Leipzig, East Germany In comparison with other skeletal muscles oculorotatory muscles exhibit a very high speed of contraction. The quick twitch response and the low twitch-tetanus ratio assumes a very short active state with a steep decay. At 35 ° C and 25 ° C isometric twitches and tetani of normal and denervated eye

Fish muscle muscles of the rabbit were recorded. Unfused tetani were analysed to estimate the decay of the active state. Depending on the period after denervation the decay of the active state is prolonged. The diminished maximum tension after denervation is likely to be caused by a diminution of the amplitude of the active state. The influence of different factors on the active state of normal and denervated eye muscles is discussed. Probably the results are explicable by a diminished Ca 2+ reaccumulation in denervated eye muscle fibres.

FISH MUSCLE Histochemical pattern in the red and white muscle of fishes and some biochemical data on myofibrillar ATPase E. C a r p e n ~ 1, A. V e g g e t t i 2, F. Mascarello 3 a n d P. Scapolo 2

llstituto di Biochimica dell'Universit?~ di Bologna 2Istituto di Anatomia degli Animali Domestici con Istologia ed Embriologia delle Universit~ di Bologna e 3de Milano, Italy

#~

One of the most frequently studied fish proteins is myosin, of which different forms occur in different histochemical fibre types. Biochemical differences have been shown to occur between the myosins of fish adapted to different temperatures [Penney et al., I ESCPB Conf. Anim. Envir. Fitness, Gilles, Oxford, Pergamon Press (1979)]. The present report describes the results of a histochemical investigation (mainly using the ATPase reaction according to Padykula & Herman LJ- Hislochem. Cytochem. 3, 170 (1955)] and Guth & Samaha [Exptl Neurol. 25, 138 (1969)] to see if there is any connection between the muscle fibre type composition and the habitat. The fish investigated were from fresh, salt and brackish water. Some species were investigated at intervals throughout the year, and the stability of the Ca2+/Mg2+-myofibrillar ATPase was investigated for Ictalurus melas and Mugil chelo. We found that although the myosin ATPase reaction pattern varies from species to species, this does not seem to correlate with the habitat. The acid-labile myosin ATPase activity typical of fast twitch fibres is present in the white fibres of most species. However in the white muscle of Gobius paganeUus the enzyme activity is stable to both acid and alkali. It has been possible to demonstrate a seasonable variation in the fibre pattern of the white muscle of the mugilidae: in late summer small diameter fibres with acid- and alkali-resistant ATPase activity appear, scattered between the fibres of the white muscle. In autumn, after all the small fibres have reached the diameter typical of white fibres, no small fibres appeared until the following summer [Carpen6 & Veggetti, Experientia 37, 191 (1981)].

Biochemical assay of Ca2+/Mg2+-myofibrillar ATPase from mullet and black bullhead, revealed a much greater thermostability in the black bullhead than in the mullet. This is consistent with the high temperature that the black bullhead can tolerate.

Contractile properties of a white and a red fibre type of the m.hyohyoideus of the carp

(Cyprinus carpio ) H. L. M. G r a n z i e r 1, J. W i e r s m a 2, J. W. M. O s s e 1, H. A. A k s t e r 1 a n d W. M. C. M. v a n Dommelen 1

1Department of Experimental Animal Morphology and Cell Biology, Marijkeweg 40, Agricultural University, Wageningen, The Netherlands 2Department of Animal Physiology, Haarweg 10, Agricultural University, Wageningen, The Netherlands Isometric contraction times were measured for small fibre bundles, isolated from the m.hyohyoideus superior, containing mainly (> 90% cross-section surface) pink fibres (a white type) and from bundles isolated from the m.hyohyoideus inferior, which contained mainly red fibres. Fibre types were characterized on the basis of immune histochemical reactions and histochemical reactions for ATPase and succinate dehydrogenase. Both fibre types have multiterminal innervation. Stimulation of the nerve was performed at the fibre length giving optimal twitch tension. Pink fibres have shorter twitch contraction times than red fibres (32 ms for pink and 60 ms for red) and a shorter half relaxation time (37 ms for pink and 106 ms for red). Pink fibres showed contraction summation at a stimulation frequency of 5-7 Hz, the tetani became fused at 60-75 Hz and maximal tetanic tension was at 75-150 Hz. For red fibres these values were respectively: 1-2 Hz, 40-50 Hz and 175-300 Hz. Maximal tetanic tension was similar for both fibre types: 11.8 N cm -2 for the pink fibres, 11.2 N cm -2 for the red fibres. The twitch-tetanus ratio was 0.42 for pink fibres and 0.27 for red fibres. Repeated tetanization of the pink and red fibres showed that the red fibres had a greater resistance to fatigue.

Fish muscle Differentiation of the sarcoplasmic proteins of white skeletal, cardiac and swim bladder muscles of the toadfish, Opsanus tau G. H a m o i r a n d N. G e r a r d i n - O t t h i e r s

Laboratoire de Biochimie musculaire, Institut de Chimie, UniversitO de Liege, Sart Tilman, B-4000 Liege, Belgium The swimbladder muscle of Opsanus tau differs from the parietal white muscle by a two to threefold higher parvalbumin content and a peculiar parvalbumin distribution [Hamoir et al., J. molec. Biol. 143, 155-60 (1980)]. Trunk, swimbladder and heart muscles differ also by their myoglobin content of 0.1, 0.6 and 8 mg g muscle -1 while a unique electrophoretic b a n d is observed for creatine kinase even in heart. Each muscle type has its o w n LDH pattern. Fast and swimbladder muscles contain the same isozymic group of 4 or 5 bands but the distribution is obviously different. The two slower bands are hardly visible in trunk muscle. Furthermore, another faint group of much slower isozymes occurs sometimes in the pattern of swimbladder muscle. Heart muscle, on the other hand, contains as m u c h as 10 isozymes; their separation appears, however, hindered by some adsorption. In conclusion, the superfast swimbladder muscle does not seem to rank within the fash twitch oxidative muscles.

Electrophoretic and electromyographic study of some head muscles of the c a r p B. F o c a n t , F. H u r i a u x a n d P. V a n d e w a l l e

Laboratoire de Biochimie Musculaire, Institut de Chimie, Sart Tilman, 4000 Liege, Belgium We have examined the myofibriUar proteins of the carp adductor mandibulae muscles (Ale, A1/~, A 2 and A 3) on one- and two-dimensional polyacrylamide gel electrophoresis. The percentage of red fibres of these muscles was calculated according to the proportions of white and red light chain types appearing on urea and SDS gels of their myosins. Muscles were classified from 'white' to 'red' in the following order: W(0%) ~ A 2 -~ AlCr ~ Alfl -~ A 3 ---* R(100%). Our electromyographic study revealed that adductor mandibulae A3, containing the higher amount of red fibres, was the sole muscle involved in the respiratory cycle. More careful dissection of white and red parts of each muscle (when noticeable) confirmed the marked heterogeneity of adductor mandibuiae muscles. In order to gain a better insight into the differentiation of fish head muscles myofibrillar proteins, we carried out biochemical and physiological investigations on these parts relating fibre type and function. Electrophoretograms as well as electromyograms show that Ale, Alfl and A 3 muscles are constituted by several parts differing by their fibre composition and their activity pattern.

123 Induced conversion of white fibres into intermediate fibres of fish myotomal muscle K. V e e k e n , W. v a n R a a r n s d o n k a n d W. v a n d e L a a r s e

Zoological Laboratory, University of Amsterdam, Plantage Doklaan 44, 1018 CN Amsterdam, The Netherlands Myotomes of teleost fishes contain three main fibre types: slow red fibres, in a thin lateral part, intermediate fast fibres in an area adjacent to the red and, in a large medial part, white fast fibres. These three fibre types are recruited successively with increasing swimming speeds. Throughout the dorsal and ventral portion of the 'white area', there are some scattered intermediate (SD-SV) fibres. The function a n d the origin of these fibres is not yet clear. They might be recruited together with the fibres of the 'intermediate area' in order to overcome the resistance of the white muscle portion w h e n the body is being bent [ van Raamsdonket aL, Acta histochem. 67, 200-16 (1980)]. This hypothesis predicts an increase in the n u m b e r of S D - S V fibres from cranial to caudal, related to the curvature of the body at normal swimming movements. This is exactly what we have found. Next we tried to induce the formation of S D - S V fibres by changing the pattern of body movements. Spinal cord lesions force the fish to make undulating movements with the anterior part of the body. This lesion induces an increase in the n u m b e r of S D - S V fibres in the cranial part of the body musculature. The results provide support for our hypothesis and they also indicate that activity training causes a change in the myofibrillar properties from white to intermediate.

AMP deaminase in goldfish muscles Aren van Waarde and Fanja Kesbeke

Department of Physiology, Zoological Laboratory, University of Leiden, P.O. Box 9516, 2300 RA Leiden, The Netherlands Goldfish muscle A M P deaminase has been studied by examination of its regulatory properties in vitro and its hmction in intact fish during anoxia and exercise. Since the goldfish myotome consists of red and white fibres, which are spatially separated from each other and are used at different swimming speeds, a distinction was made between them. The enzyme has been purified 160-190-fold from muscle homogenates by means of (NH4)2SO 4 precipitation and cellulose-P chromatography. Red muscle and white muscle contain different isozymes showing a different behaviour on cellulose-P, a different dependence of activity on pH and a different response to activators. Regulatory properties of the enzyme have been studied at 120mM KC1 and 0.1 mM AMP. ADP and ATP are activators, lowering the K m for AMP. Mg 2+ ions counteract the activating effect of adenylates. GTP and Pi are inhibitors, but their effect is d e p e n d e n t on the composition of the reaction mixture: in the presence of a physiological adenylate pool inhibition is not very strong. The

124 white muscle enzyme shows a much steeper response on changes of ADP concentration than the enzyme from red muscle, indicating that during exercise white muscle enzyme can be activated faster and more strongly than the red muscle isozyme. The main factors regulating enzyme activity in vivo appear to be the concentrations of free AMP and ADP. During exercise AMP deaminase is activated in white muscle, as indicated by a decline of the adenylate pool and an increase of IMP and NH 3. IMP and NH 3 accumulate in stoichoimetric amounts, suggesting that NH 3 does not leak out during the short (10 min) exercise period. During anoxia, AMP deaminase is activated both in red muscle and white muscle. IMP accumulation is detectable but, in the long anoxia period (12 h), NH 3 is removed by the circulation. Adenylate energy charges of muscle tissue never drop below 0.8 due to the action of AMP deaminase.

Development and differentiation of jaw muscles of fish P. M. G. Barends a n d A. J. Thiele

Department of Experimental Animal Morphology and Cell Biology, Agricultural University, Wageningen, The Netherlands The development and differentiation of the jaw adductor muscle in Barbus conchonius have been studied in order to determine the relation between fibre type differentiation and early muscle activity. The jaw adductor muscle, which functions in mouth-closing, consists of two fibre groups: one, involved during different activities of variable strength and duration and consisting largely of oxidative fibres, and the other, being active during short vigorous movements and consisting of mainly glycolytic fibres. The latter shows a high alkali-stable ATPase activity, whereas the oxidative fibres have an alkali- and acid-labile ATPase. In adult barbs, this resembles the jaw adductor muscle of the perch [Barends, Proc. Kon. Ned. Acad. WeL C 82, 147-64 (1979)]. The developing jaw muscles of larval barbs have been studied with (immune) histochemical methods from two days post-fertilization (pf) onwards. Filaments of muscle proteins are present in three-day-old and older larvae (EM). On the third day, nervous tissue and end plates are also present. From the 4th day pf onwards a distinction in (irnmune)histochemical characteristics between deeply located future-red fibres and more superficial future-glycolytic fibres is observed (Barends, van Leeuwen and Thiele, unpublished results). First movements of the mouth are observed during the third day pf, An autoradiographic study combined with an ultrastructural investigation to determine the start and time course of the differentiation of jaw muscle fibres shows only one pool of dividing myoblasts. This probably sends its mitotic products in two directions: one laterally to form glycolytic fibres, and the other medially to form oxidative fibres.

Fish muscle

Fibre types in axial muscles, the m. hyohyoideus and masticatory muscles of the carp (Cyprinus carpio L.) H. A. Akster a n d F. A. S i b b i n g

Department of Experimental Animal Morphology and Cell Biology, Agricultural University, Wageningen, The Netherlands An inventory of fibre types was made in the lateral axial muscles, the m. hyohyoideus, the epaxial muscles, the rn. sternohyoideus and in several pharyngeal bone (gill arch V) muscles. On the basis of histochemical reactions on ATPase and succinate dehydrogenase and, in the first two muscles, also on immune histochemistry [after Van Raarnsdonk et al., Acta histochem. 67, 200-16 (1980)] the following types were distinguished: three types of white fibres, (white a, white b or large pink and white c or small pink, which differ in diameter, pH stability of their ATPase and in reactivity with an antiserum), and two types of red fibres (differing in pH stability of their ATPase and in SDH activity). White fibres have more T-system/SR contact than red fibres. This parameter is supposed to be related to contraction velocity. Activity patterns during intensive respiration, food intake and mastication, and the functional roles in crushing and grinding of all but the first two muscles were previously determined by electromyography and X-ray cinematography (Sibbing, unpublished results). These muscles have multiple functions and contain several fibre types. Muscles that are mainly power supplying have homogeneous white (pink) and red parts. Muscles that have more of a guiding and steering role contain a mosaic of types. White fibres (type a) are only found in muscles that are highly (+ +) active in sudden movements requiring high force and high velocity (food intake). Red fibres, which are found in all muscles, are held to be responsible for the recorded activity during respiration [Akster & Osse, Neth. J. Zool. 28, 94-110 (1978); Barends, Proc. Kon. Ned. Acad. Wet. C 82, 147-64 (1979)]; they are employed in slow, repetitive and lasting movements. Pink fibres (type white b or white c) are found in all muscles that are highly active in mastication, a repetitive though not lasting process, requiring velocities intermediate between rapid food intake and rather slow respiratory movements.

The effects of acclimation to hypoxia on the capillary supply and mitochondrial content of tench (Tinca tinca) skeletal muscle fibres Ian A. J o h n s t o n a n d L y n n e M. Bernard

Department of Physiology, University of St Andrews, St Andrews, Fife, Scotland A quantitative analysis of muscle ultrastructure and capillary supply has been carried out on tench (Tinca tinca) acclimated for six weeks (15 ° C) to either airsaturated (P02 - 21 kPa) of oxygen deficient (Po2 - 1.7 kPa) water. Acclimation to reduce oxygen levels resulted in a significant reduction in the fraction (FV) of slow fibre volume occupied by

125

Fish muscle mitochondria (22.9-15.0%) together with parallel changes in capillary supply (1.8 ± 0.1 to 1.0 -+ 0.07, capillaries/fibre: mean +- S.E.M.). The majority of slow muscle mitochondria occur in the subsarcolemmal zone. Intermyofibrillar mitochondria (FV: 4.4% normoxic; 1.2% hypoxic) were affected by low oxygen to a greater extent than subsarcolemmal populations (FV: 18.5% normoxic, 13.8% hypoxic). Mitochondria from hypoxic fish often appeared swollen with a reduction in the complexity of cristae. The capillary surface (~m) supplying 1pro 3 of fibre volume decreased significantly (0.032--0.018) following acclimation to hypoxia while the ratio of capillary surface to mitochondria volume remained approximately constant. Mitochondrial FV (4.5-1.8%) and the number of capillaries/fibre (0.8-0.2) decreased to a somewhat greater extent in the fast muscles of hypoxic acclimated fish. Fibre size was unchanged and actively differentiating fibres were observed in both groups of fish. The results demonstrate the plasticity of fish muscle ultrastructure in relation to environmental oxygen availability. (Supported by a grant from the N.E.R.C.)

Molecular plasticity in the myofibrillar protein complex of the m u m m i c h o g (Fundulus

heteroclitus ) I. A. J o h n s t o n , B. D. Sidell, T. S. M o e r l a n d a n d G. G o l d s p i n k

Department of Physiology, Universityof St Andrews, Fife, Scotland; Department of Zoology, University of Maine, Orono, Maine, U.S.A. and Department of Zoology, University of Hull, England Mummichogs from salt marshes of the Atlantic coast of Maine (U.S.A.) experience a mean seasonal temperature range of - 1 ° C to +15 ° C. However, during summer tidal cycles they maoyexperience rapid temperature changes between 15 C and 30 ° C. Observations of animals in the wild suggest that swimming capability is conserved during acute temperature fluctuations which would substantially impair contractile function in other fishes. Myofibrils were isolated from the fast epaxial musculature and ATPase activity determined in a medium of 40ram imidazole pH 7.2 (25 ° C), 50 mM KC1, 5 mM ATP, 6 mM MgC12, 5 mM EGTA 0.4rag protein -1 ( / = 0 . 1 2 4 ) in the presence (pCa 7.15) and absence (pCa 5.15) of 5 mM CaC12. ATPase achwhes " " " were > 90 °o ~ Ca 2+-sensitive over the temperature range 0-35 ° C. Arhenius plots of Mg2+/Ca2+-myofibrillar ATPase activity showed a discontinuity in slope at - 1 2 ° C. Values for activation enthalpy (AH*) of the ATPase were 28000 kcal tool -1 in the range 0-12.5°C and 12 000 cal tool -1 between 12.5-35 ° C. The pCas required to give half maximal ATPase activity were 6.58, 6.25 and 6.20 at 5, 15 and 25 ° C, respectively. Thermal denaturation of Ca 2+-sensitivity and ATPase activity proceed essentially in parallel. At

0.5 mgm1-1 in standard incubation medium the temperature required to denature 50% of activity over 30 min is 40-41 ° C. Identical results were obtained for groups of fish acclimated for six weeks to either 5 ° C, 15°C or 25 ° C. The higher temperature dependence and relatively low ATPase activity at very cold temperatures suggests a relatively torpid state for overwintering fish. Compared to other fishes previously studied, results with mummichogs indicate a pronounced plasticity in both Ca 2+ regulatory and catalytic activity of the myofibrillar complex over the range of 12-35 ° C. These factors apparently obviate the need for acclimatory modifications of the contractile proteins. [Supported by grants from N.A.T.O., N.S.F. (U.S.A.) and S.E:R.C. (U.K.).]

The origin and differentiation of red and white muscle fibres in the sturgeon (Acipenser

stellatus ) Per R. Flood a n d H a r a l d Kryvi

Institute of Anatomy, University of Bergen, 5000 Bergen, Norway As part of a larger research programme on the ontogo eny and phylogeny of distinct skeletal muscle fibre types we have examined the origin of red and white muscle fibres in the sturgeon Acipenserstellatus. Fixed and partly dehydrated embryos at different developmental stages, were kindly supplied by Dr Guljaev and Dr Sakharov at the Institute of Developmental Biology in Moscow. Further processing for light and electron microscopy was made in Bergen. Muscular contractions first appear 1½ days before hatching at stage 30 and the somite at this stage consists of a thick medial layer of myogenic cells and a thin lateral layer of probably dermatogenic cells. The myogenic layer consists of flattened multinuclear lamellae stacked on top of each other in the ventro-dorsal direction. The lamellae are wedge-shaped with their bases alternating in the medial and lateral direction throughout the stack. Myofibrillogenesis starts along the sides of the medially based wedges. The medial portion of the laterally based wedges are often so thin that the electron microscope must be used to trace them to the medial border of the somite. At stage 31, myofibrils are also present along the lamellae with lateral bases. At hatching (stage 36), the lateral wedges have withdrawn most of their apical portion and acquired a deeper stainability than the medial wedges. Four days after hatching (stage 40), the lateral 'wedges' constitute a single superficial layer of cylindrical fibres with typical red or aerobic characteristics. The medially based wedges have split off their lateral portions to form an intermediate layer of cylindrical fibres,: whereas their medial portions are still lamellar in shape. This medial layer reveals typical white or anaerobic ultrastmctural characteristics. Fifteen days after hatching the superficial red fibres start to split. At 22 days after hatching mostly two

126

Fish muscle

layers are found, and at 48 days 3-5 layers of red fibres are found. During the same period the deep white lamellae have continued to split off cylindrical fibres from their lateral and medial borders until, at 48 days after hatching, all the lamellae are transformed into irregular cylindrical fibres. The relative volume fraction of mitochondria and myofibrils in the red and white fibres has been measured at distinct developmental stages and differs for the two fibre types from stage 38 (2 days after hatching) onwards. However, the mature-like values may not be reached until the animal is about six months old.

AUTHOR

We conclude that presumptive red and white fibres originate from alternating cells throughout a singlelayered stack of fused myoblasts in the myotome as soon as muscular contractions appear (1.5 days before hatching) and that the classical cytological differences between red and white fibres start to develop at the moment of hatching. Further we conclude that the number of both red and white fibres increases after hatching by splitting rather than by myoblast fusion. Intermediate fibres seem to originate by splitting from pre-existing embryonic white fibres.

INDEX

Akster, H. A., 122, 124 Asmussen, G., 122 Barends, P. M. G., 124 Beier, N., 118 Berchtold, M. W., 114 Bernard, L. M., 124 Bernengo, J. C., 116 Cardinaud, R., 116 Carpene, E., 122 Celio, M. R., 114 Chin, T. K., 118 Clancy, M. J., 115, 116 Conneely, O. M., 120 Dancker, P., 117 De Beer, E. L., 119 D'Haese, J., 117 Diegenbach, P. C., 113 Ditgens, A., 117 Flood, P. R., 125 Focant, B., 123

Gerardin-Otthiers, N., 123 Ghins, E., 115 Gillis, J. M., 117 Godfraind-de Becker, A., 117 Goldspink, D. F., 113, 114, 119 Goldspink, G., 125 Granzier, H. L. M., 122

Kesbeke, F., 123 Kil, P. J. M., 121 Kodama, T., 121 Kordowska, J., 116 K6ssler, G., 121 Krajewski, K., 116 Kryvi, H., 125 Kfichler, G., 121 Kulesza-Lipka, D., 116

Reist, U., 118 Roche, J. F., 116 Rowe, A. J., 118 Rowlerson, A., 118 Scapolo, P., 122 Schiereck, P., 121 Schnackerz, K. D., 118 Sibbing, F. A., 124 Sidell, B. D., 125

Lefevre, J., 117 Hamoir, G., 123 Lester, J., 115, 116 Headon, D. R., 119, 120 Lewis, S. E. M., 119 Heilmeyer, L. M. G., 118 Heizmann, C. W., 114 McGrath, J. A., 114 Hooper, A. C. B., 113 Makinose, M., 120 Howland, H., 118 Marechal, G., 115 Huriaux, F., 123 Mascarello, F., 122 Moerland, T. S., 125 Jakubiec-Puka, A., 116 Molloy, M. A. N., 120 Jenny, E., 118 Johnston, I. A., 124, 125 Osse, J. W. M., 122

Van der Laarse, W. J., 113, 123 Vandewalle, P., 123 Van Dommelen, W. M. C. M., 122 Van Raamsdonk, W., 123 Van Waarde, A., 123 Veeken, K., 123 Veggetti, A., 122

Kelly, F. J., 114

Wiersma, J., 122

Quinn, P. J., 119

Thiele, A. J., 124